Environment responsive gelling copolymer

ABSTRACT

The invention relates to a gelable copolymer composition comprising a copolymer in a solvent. The copolymer has the structure A(B)n, wherein the core (A) is soluble in the solvent, the arms (B) are convertible between soluble and insoluble in the solent depending on an environmental condition, and n&gt;1. The composition forms a gel under environmental conditions in which B is insoluble, through formation of B aggregates. Block copolymers comprising polyethylene glycol (PEG) and poly(N-iso-propylacrylamide) (PNIPAAm) having a liquid form at ambient temperature under aqueous conditions and a gel form at body temperature are disclosed. Copolymer compositions according to the invention can be used to form in situ implants useful in slow-release formulations of biologically active molecules.

FIELD OF THE INVENTION

The present invention relates generally to copolymers. Moreparticularly, the present invention relates to polymers havingproperties responsive to environmental changes.

BACKGROUND OF THE INVENTION

Block and graft copolymers are used for a variety of physiological andindustrial applications. The solubility of a copolymer in a particularsolvent depends inter alia on the characteristics of the monomericcomponents incorporated into the copolymer.

Polymers capable of gelation induced by environment changes are known.Solvent-induced gelation has also been exploited as a mechanism forproducing in situ gelable materials. The solvent-induced gelationconcept employs a polymer that is soluble in a non-aqueous solvent, butinsoluble in water. When a non-aqueous solution of such a polymer isinjected into an aqueous environment, the non-aqueous solvent isexchanged for water and the polymer precipitates, forming a solid massin situ. Solvent-induced gelation systems have the disadvantage that theinitial fluid form of the polymer is formed in a solvent other than thesolvent in which the gel eventually forms. U.S. Pat. No. 5,744,153 (Apr.28, 1998) and U.S. Pat. No. 5,759,563 (Jun. 2, 1998), both to Yewey etal., describe a composition for in situ formation of a controlled drugrelease implant based on the solvent-induced gelation concept.

A series of patents to Dunn et al. also describe a solvent-induced gelcomposition (U.S. Pat. No. 5,739,176 issued Apr. 14, 1998; U.S. Pat. No.5,733,950 issued Mar. 31, 1998; U.S. Pat. No. 5,340,849 issued Aug. 23,1994; U.S. Pat. Nos. 5,278,201 and 5,278,204 both issued Jan. 11, 1994;and U.S. Pat. No. 4,938,763 issued Jul. 3, 1990). The compositionincludes a water-insoluble polymer and a drug solubilized in an organicsolvent carrier. When the composition is injected into a physiological(aqueous) environment, such as a human subject, the polymer precipitatesto form a solid mass. Solvent-induced gel compositions have thedisadvantage that an organic solvent is injected into a subject merelyto carry the polymer and drug in a liquid form. Thus, the organicsolvent must subsequently be metabolized or cleared by the body.

Self-assembling hydrogels have been receiving increasing attention inthe last few years, both for their intrinsic scientific interest, andfor their potential clinical and non-clinical applications. A number ofelegant mechanisms for self-assembling hydrogels have been proposed.Nagahara et al. showed that gels can be formed by complexation betweencomplementary oligonucleotides grafted onto hydrophilic polymers(Polymer Gels and Networks, 4: (2) 111-127, 1996). Miyata et al.prepared antigen sensitive hydrogels based on antigen-antibody binding(Miyata et al., Macromolecules, 32: (6) 2082-2084, 1999; Miyata, Nature,399: (6738) 766-769, 1999). Petka et al. illustrated a gelationmechanism using triblock copolymers containing a central hydrophiliccore and terminal leucine zipper peptide domains (Science, 281: (5375)389-392, 1998). The terminal domains form coil-coil dimers or higherorder aggregates to provide crosslinking when cooled from above itspH-dependent melting point. Thermoreversibility was demonstrated withsome hysteresis due to the slow kinetics of coil-coil interactions.

Triblock copolymers having a central hydrophobic poly(propylene oxide)(PPO) segment and hydrophilic poly(ethylene oxide) (PEO) segmentsattached at each end are commercially available. The aqueous solution ofthese triblock copolymers (PEO-PPO-PEO) have a fluid consistency at roomtemperature, and turn into weak gels when warmed to body temperature byforming oil-in-water micelles in aqueous solution. The gelation of thepolymer is believed to occur via the aggregation of the micelles(Cabana, et al., J. Coll. Int. Sci., 190 (1997) 307).

A group led by S. W. Kim have reported the development ofthermosensitive biodegradable hydrogels (Jeong et al., J. ControlledRelease, 62 (1999) 109-114; Jeong et al., Macromolecules, 32: (21)7064-7069, 1999; Jeong et al., Nature, 388 (1997) 860-862). Thesehydrogels are block copolymers of PEO and poly(L-lactic acid) (PLLA) ineither a di-block architecture PEO-PLLA, or a tri-block architecturePEO-PLLA-PEO. They also report triblock copolymers of poly(ethyleneoxide) and poly(lactide-co-glycolide) (PLGA) having the architecturePEO-PLGA-PEO. Aqueous solutions of these polymers were reported toundergo temperature-sensitive phase transitions between fluid solutionand gel phases. In aqueous solution, these polymers form micellescomposed of hydrophobic cores (either PLGA or PLLA) and hydrophilicsurfaces (PEO). Gelation is believed to be due to the aggregation ofmicelles driven by hydrophobic interactions. This group has alsodiscussed the synthesis of PEO copolymers in multi-armed star shapedarchitectures having polycaprolactone (PCL) or PLLA chains attached tothe PEO arms.

Another class of in situ gelable materials is based on polymers madefrom proteins, or “protein polymers”. Cappello, et al. (J ControlledRelease 53 (1998) 105-117) reported gel-forming block copolymers basedon repeating amino acid sequences from silk and elastin proteins. Whenheated to body temperature, the proteins self-assemble via a hydrogenbond mediated chain crystallization mechanism to form an irreversiblegel. The gelation occurs over a relatively long time period of more than25 minutes.

Although a variety of gelling or precipitatable polyethyleneglycol/poly(N-isopropylacrylamide) copolymers have been synthesized,none was designed and synthesized with in situ gelation applications inmind. See, for example Yoshioka et al., J. M. S Pure Appl. Chem., A31:(1) 109-112, 1994; Yoshioka, J. M. S. Pure Appl. Chem., A31: (1)113-120, 1994; Yoshioka, J. M. S Pure Appl. Chem., A31: (1) 121-125,1994; Kaneko, Macromolecules, 31: 6099-6105, 1998; Topp, et al.,Macromolecules, 30: 8518-8520, 1997; and Virtanen, Macromolecules, 33:336-341, 2000.

Topp et al. disclose block copolymers of PEG and PNIPAAm having thestructure of either PNIPAAm-PEG or PNIPAAm-PEG-PNIPAAM which formspherical micelles in aqueous solution (Macromolecules, 30: 8518-8520,1997). The block copolymers were synthesized by the Ce⁺⁴ initiatedattachment of NIPAAm monomers onto the hydroxyl terminals of PEG chains.It was shown that as PNIPAAm segments grew in length during synthesis,micelles having a PNIPAAm core and PEG corona were formed, and thepolymerization of PNIPAAm chains continued in the core of the micelles.The copolymers formed by Topp et al. are of a form appropriate for usein a surfactant composition for drug loaded micelles. However, micellesare isolated entities having no load bearing characteristics, do notform gels, and the formation of micelles is associated with a dilutesolution state.

The block copolymers formed by Topp et al. consisted of compositionswith PNIPAAm to PEG mass ratios (M_(n,PNIPAAm)/M_(n,PEG)) ranging fromabout 0.14 to 0.48, and they found that block copolymers with aM_(n,PNIPAAm)/M_(n,pEG) ratio exceeding ⅓ show aggregation in water attemperatures below the lower critical solution temperature (LCST) atwhich a solubility change occurs, and thus are less useful for micelleformation than copolymers with ratios less than ⅓.

There is a need for a gelable polymer that is responsive toenvironmental changes other than solvent exchange. Further, there is aneed for a gelable polymer composition capable of reversibly forming astrong gel.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a gelable polymerand composition which obviate or mitigate at least one disadvantage ofprevious gelable polymer systems.

In a first aspect, the present invention provides a gelable compositioncomprising a copolymer and a solvent, the copolymer having the structureA(B)n, wherein A is soluble in the solvent, B is convertible betweensoluble and insoluble in the solvent depending on an environmentalcondition, and n is greater than 1, the composition being convertiblefrom liquid to gel under an environmental condition where B isinsoluble. Optionally, the composition according to the invention mayadditionally comprise an architecturally different copolymer, so thatn_(avg) for all copolymers present in the composition is greater than 1.

In a further embodiment, there is provided a gelable copolymer havingthe structure A(B)n, wherein A is soluble in a desired solvent, B isconvertible between soluble and insoluble in the desired solventdepending on an environmental condition, n is greater than 1, and A(B)nforms a gel in the solvent under an environmental condition where B isinsoluble.

In further aspect, the present invention provides a gelable copolymercomprising a core selected from compounds soluble in a desired solvent,and arms selected from compounds convertible between soluble andinsoluble in the desired solvent depending on an environmentalcondition, wherein the copolymer forms a gel in the solvent under anenvironmental condition where the arms are insoluble.

The invention further relates to an in situ forming implant comprising agelable composition as described above, wherein the solvent is aqueousand the environmental condition comprises heating to a temperaturebetween ambient temperature and body temperature.

The invention also provides a process for forming a gelable compositioncomprising the steps of: (i) forming a copolymer having the structureA(B)n, wherein A is soluble in a solvent of interest, B is convertiblebetween soluble and insoluble in the solvent depending on anenvironmental condition, and n is greater than 1; (ii) solubilizing saidcopolymer in the solvent in an amount adequate to convert thecomposition from liquid to gel under an environmental condition where Bis insoluble.

According to an embodiment of the invention, there is also provided aprocess for forming an in situ forming implant comprising a gelablecomposition as described herein, wherein the solvent is aqueous and theenvironmental condition comprises heating to a temperature betweenambient temperature and body temperature.

Advantageously, the gel-forming polymer according to the invention doesnot require a solvent exchange to convert between a fluid solution and agel. Thus, the polymer can be prepared and utilized in a single solventsystem.

Other aspects and features of the present invention will become apparentto those skilled in the art upon review of the following description ofspecific embodiments of the invention in conjunction with theaccompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures, wherein:

FIG. 1 is a schematic diagram of block copolymer architectures A(B)₂,A(B)₄ aid A(B)₈, and graft copolymer architectures A(B)₂ and A(B)₃,according to the invention, shown here for comparison purposes withpolymer AB.

FIG. 2 is a schematic diagram of copolymer architectures A(CB)₂ andA(CB)₄ according to the invention.

FIG. 3 illustrates an A(B)₄ polymer of PEG and PNIPAAm in aqueoussolution. Picture A illustrates a 20% wt A(B)₄ solution at 25° C., whilepicture B illustrates a 20% wt A(B)₄ gel at 37° C.

FIG. 4 illustrates that for a 20% solution of A(B)₄ the onset ofincrease in the elastic and loss moduli, shown as (A) temperature sweepof oscillatory measurement, occurs at a temperature between the onsetand peak of the endotherm as detected by (B) DSC temperature scan.

FIG. 5 shows the superposition of the DSC scans for multiple cycles forboth (A) the four-arm polymer A(B)₄ and (B) the eight-arm polymer A(B)₈,both at 20% wt in water (2° C./min for 30 cycles), illustrating the fullthermal reversibility of copolymers according to an embodiment of theinvention.

FIG. 6 illustrates parameters relating to the rheological behavior ofcopolymers. (A) Elastic Modulus vs. Oscillatory Stress; (B) OverallModulus vs. Oscillatory Stress; and (C) tan delta vs. OscillatoryStress. The tests were conducted at a frequency of 1 Hz.

FIG. 7 illustrates thermal transition (DSC scans) of compositionscontaining 50/50 copolymers of PEG/PNIPAAm according to the invention atvarious concentrations. Comparative example (a) AB is shown relative tothe inventive compositions containing (b) copolymer A(B)₂, (c) copolymerA(B)₄ and (d) copolymer A(B)₈.

FIG. 8 provides phase diagrams PEG-PNIPAAm copolymers in (a) distilledwater, (b) 157 mM NaCl solution, (c) F-12K cell culture media, and (d)extra cellular solution. C_(min) is the minimum gelation concentration,below which no gel forms over the range of temperatures investigated.

DETAILED DESCRIPTION

The invention relates to a gelable copolymer, and to a copolymercomposition that undergoes structural changes in response to changes inthe environment. Within the composition, the inventive copolymerundergoes a phase transition from liquid to gel in response to changesin an environmental parameter such as, for example temperature, pH,ionic strength of the composition, or combinations of these parameters.

The mechanism of environment responsive gel formation according to theinvention has not been observed or described previously. The inventivepolymer A(B)_(n) undergoes gel formation under specific environmentalconditions as a result of environment-sensitive aggregation of the arms(B) of the copolymer. The aggregates of arms (B) thus form physicalcrosslinks between the core component (A) of the copolymers to createthe gel structure when the environmental conditions are those underwhich the arm component (B) is insoluble. The copolymer compositionreadily converts between a liquid state (when solubilized either inaqueous or non-aqueous solvents) and a gel state when subjected tochanges in the environmental conditions.

According to one embodiment, the inventive copolymer readily dissolvesin water at room temperature to form a low viscosity solution, butbecomes a gel at a temperature just below body temperature. Thecopolymer contains an unresponsive core (A) to which a varying number ofenvironment-responsive arms (B) are attached. Thus, the copolymer has ageneral structure A(B)n. The arms (B) can be attached at any point alongthe core (A), provided the arms are accessible to the arms of othermolecules for intermolecular aggregation upon changes in environmentalconditions. For example, the arms may be attached to the ends of thecore, thus forming a block copolymer, or may be attached along the chainof the core, thus forming a graft copolymer. FIG. 1 diagrammaticallyillustrates two-arm, four-arm and eight-arm block copolymer structuresA(B)₂, A(B)₄ and A(B)₈, and graft copolymer structures A(B)₂, A(B)₃,with comparison to structure AB.

The core (A) may be a homopolymer or a copolymer, either linear orbranched, and is chosen so that the core (A) itself is soluble in theselected solvent over the range of environmental conditions of interest.The arms (B) are chosen such that B itself would switch between beingsoluble and insoluble in the selected solvent between the environmentalconditions of interest. When the core and arms are incorporated into acopolymer of structure A(B)n, the copolymer is soluble in the selectedsolvent in conditions under which the arms are soluble. However, when anenvironmental condition is changed to a condition under which the arms(B) themselves would be insoluble, the B component of the copolymerprecipitates to form aggregated domains with B components of adjacentcopolymers. The aggregated B components are linked by A segments since Band A components are covalently linked within a copolymer molecule.Thus, a three dimensional gel structure is formed containing many Asegments connected via physical crosslinks of B aggregated domains.

In the resulting gel, the inventive copolymer incorporates anequilibrium quantity of solvent due to the compatibility between core Aand the solvent, thereby forming a solvent-containing gel.

According to one embodiment of the invention, PEG is used as core A,poly(N-isopropyl acrylamide) (PNIPAAm), a temperature responsivepolymer, is used for arms B. Copolymers are formed with varying numbersof PNIPAAm arms. These copolymers are water soluble at room temperature,forming low viscosity liquid aqueous solutions. However, upon heating,the copolymers rapidly and reversibly form strong gels (in less than aminute), exhibiting little syneresis.

The gelable composition according to the invention may contain mixturesof A(B)n copolymers that contain different A components, different Bcomponents, or have different n, or any combination thereof. In thisway, mixtures can be used to optimize gelation kinetics or to achievegel properties desirable for a particular application.

The Core. The core (A) is chosen such that, on its own, the core (A) issoluble in the selected solvent over the range of environmentalconditions of interest. Thus, the core may be selected fromhomopolymers, or the core may itself be a copolymer (random, block orgraft), either linear or branched, provided that A is soluble over therange of environmental conditions of interest.

Core (A) may either be provided as a stable compound or as a degradablecompound. In the case where the core is degradable, the copolymer orcopolymer composition degrades over time under appropriate conditions.For example, if the core is biodegradable in a physiological system,eventually the polymer structure will break down, resulting in releaseof the arms, and ultimately removal of the copolymer structure from thephysiological system.

A number of possible cores (A) can be used according to the invention.The core may be selected from any synthetic, natural or biologicalpolymers, including but not limited to polyethylene glycol (PEG) ofvarying molecular weights and degrees of branching, polyvinylpyrrolidone, polyvinyl alcohol, polyhydroxyethylmethacrylate, andhyaluronic acid. Optionally, the core can have reactive groups at avariety of positions along or within its structure.

The Arms. The arms (B) are chosen such that B itself converts betweenbeing soluble and insoluble in the selected solvent when exposed to theenvironmental condition of interest.

The arms B may be selected according to an environment responsivenesssuited to the intended application of the invention. For example, for insitu clinical applications, water-solubility under ambient conditionsand aggregation under physiologic conditions is a desirable property ofB. The environmental condition triggering the switch between ambient andphysiological conditions may be selected from, but is not limited to,temperature, pH, ionic strength, and combinations thereof.

A number of choices for the arms (B) of the copolymer exist, including,but not limited to poly-N-isopropyl acrylamide (PNIPAAm), which is atemperature responsive polymer, hydroxypropylmethyl cellulose and othermethyl cellulose derivatives, poly(ethylene glycol vinyl ether-co-butylvinyl ether), polymers of N-alky acrylamide derivatives, poly(aminoacid)s or peptide sequences such as silk and elastin peptides,poly(methacryloy L-alanine methyl ester), poly(methacryloy L-alanineethyl ester). Nitrocellulose may be used as arms (B), for example whenethanol is used as solvent. Nitrocellulose in ethanol is known to formgel upon warming (Newman et al., J. Phys. Chem. 60:648-656, 1955).

Arms (B) may be formed from a copolymer, for example a copolymer ofvinyl ether of ethylene glycol and butyl vinyl ether, which may be usedin an aqueous solvent system. For a copolymer, the LCST beyond which apolymer changes solubility, depends on the mole ratio of the constituentcomponents. In the examples given by Kudaibergenov et al. (Macromol.Rapid. Commun, 16: 855-860, 1995), the LCST values range from 20° C. to90° C. over a mole ratio range of 72:28 to 95:5.

Arms (B) may be formed from poly(methacryloyl-DL-alanine methyl ester)or derivatives thereof. In the paper by Ding et al. (Radiat. Phys.Chem., 42 (4-6): 959-962, 1993), the LCST of the examples given arebetween 20° C. to 40° C. The gel swells at low temperature (i.e., 0° C.)and starts to de-swell upon warming to 20° C. or above.

Further, the arms (B) may be formed of methyl cellulose or derivativesthereof. Depending on specifics of the chemical composition, especiallythe degree of methylation, methyl cellulose and its derivatives werereport to have a LCST in the range of 40° C. to 70° C. (Nishimura etal., Macromol. Symp., 120: 303-313, 1997).

The arms (B) may be attached to the unresponsive core (A) at anylocation on the core, as long as the arms remain accessible to the armsof adjacent copolymer molecules, as part of the inventive composition.This structure allows for intermolecular aggregation of arms (B) whenthe environmental condition is altered such that B itself would becomeinsoluble in the selected solvent. For example, arms B may be positionedat the ends of the core, thus forming a block copolymer, or along thechain of the core thus forming graft copolymers.

As used herein, the structure “A(B)n” denotes a copolymer having arms(B) positioned on the core (A) in any manner, so as to form a block orgraft copolymer. Arms (B) may be located at one or more ends of A,forming a block or star copolymer configuration, or may be located alongthe length of the core, thereby forming a graft copolymer, with Bpositioned as “brushes” along the core, or may be positioned randomlyalong the core, provided the arms are accessible for aggregation withthe arms of adjacent molecules.

Further, as the structure “A(B)n” is understood to mean that A and B arepresent in the specified ratio within a given molecule, but that thecovalent bond between A and B may also comprise an additional component,resulting in A and B being covalently linked through such an additionalcomponent. An example wherein the additional component is a reactivespacer is described in more detail below.

The number of arms (B) attached to the core (A) is selected such that nof A(B)n is a number which is larger than, but not equal to one. For anygiven copolymer molecule, n is an integer greater than 1. Thus, theratio of arms to core in the architecture of any given copolymermolecule is 2:1, (n=2) or greater. For example, the ratio of arms tocore can be 4:1 (n=4) or 8:1 (n=8). The number of arms is not limited,provided that core is of adequate size to accommodate the selectednumber of arms, while still allowing the arms of one copolymer moleculeto access the arms of an adjacent copolymer molecule when in solution.The selection of the number of arms may also depend on the desiredproperties of the gel, for example, to achieve a stronger or weaker gel,the number of arms may be adjusted.

The gelable composition formed according to the invention may becomprised of a plurality of different copolymers. Taking into accountthe proportions of different copolymer architectures within thecomposition, an average A(B)n can be determined for the composition. Inthis case, the average n (n_(avg)) must be greater than 1, butnon-integer values of n_(avg) are possible for any particular gelablecomposition. For example if the composition contains a mixture ofcopolymers of varying architectures, such as 50% copolymer AB and 50%copolymer A(B)₂, the n_(avg) of the composition is 1.5. In the inventivecomposition, n_(avg)>1, taking into account all forms of A(B)ncopolymers in the composition. For any individual copolymer moleculewithin the composition, n is an integer number, as described above. Incompositions which contain a mixture of copolymers, it is possible tohave a gel-forming composition comprising some copolymer molecules withn=1, some with n=4, etc. In order for such a composition to be gelableaccording to the invention, n_(avg) should be adequately greater than 1,so that enough copolymer molecules with n>1 are present in thecomposition to allow formation of the gel network. In this way,copolymer molecules having the structure AB (n=1), which would notordinarily form a gel with other AB copolymers, can become part of thegel network by having their single arm segment incorporated into theaggregates formed by the molecules having n>1.

Reactive Spacers. Reactive spacers “C” may be present between core A andarms B, thereby forming a copolymer of the generic structure A(CB)n. Itis understood that A(CB)n is a variant or embodiment of A(B)n, as thestructure A(B)n is understood to mean that A and B must be present inthe specified ratio, but that the covalent bond between A and B may alsocomprise an additional component, resulting in A and B being covalentlylinked through component C.

FIG. 2 illustrates two-arm and four-arm copolymer structures withreactive spacers C. As can be seen in FIG. 2, when a reactive spacer Cis present between A and B, the basic structure of A(B)n is met, andmerely includes an additional component C within the covalent bondsbinding A to B. In the embodiment of A(CB)n, two covalent bonds bind Ato B, specifically, the bond between A and C, and the bond between C andB.

Reactive spacers C may be incorporated to allow cleavage of thecopolymer, for such purposes as for rendering the copolymer degradableunder desired conditions. Reactive spacer C may degrade via any suitablereaction, including but not limited to chemical reactions, biochemicalreactions, enzymatic degradation, or photo-induced reactions. In thecase where a reaction of the reactive spacers results in cleavage of thecopolymer, as C degrades, A(CB)n is split into individual A and Bcomponents. In the context of a physiological application, if core A andarms B are of low enough molecular weight, they can be cleared from thesite and removed from the body via renal clearance.

Biologically Active Molecules. A biologically active molecule may beincluded in the invention either through covalent attachment of themolecule to the structure of the copolymer or by including the moleculein a copolymer composition. In the case where the biologically activemolecule is included in the copolymer composition, but not incorporatedinto the copolymer itself, the biologically active molecule is optimallyselected from those having some degree of solubility in the desiredsolvent.

According to an embodiment wherein the biologically active molecule D isattached to the copolymer, it may be bound to either the core (A) or thearms (B) in such a way that the attachment allows release of thebiologically active molecule D from the copolymer. For example, acovalent attachment of D to A may occur via a degradable spacer, such asC, described above.

As with the introduction of reactive spacer (C) in the copolymer,introduction of biologically active molecule D, with or without spacerC, is considered an embodiment of A(B)n. It is understood that D may becovalently attached to either A or 13, and a copolymer polymer so formedwould meet the requirement structure of A(B)n. The structure A(B)n isunderstood to mean that A and B must be present in the specified ratio,but that the covalent bond between A and B may also comprise anadditional component such as D, through which the covalent attachment ofA and B, may be indirectly achieved.

According to a further embodiment of the invention, biologically activecomponents may be included in the polymeric composition formed accordingto the invention, but without any covalent link to the polymer itself.Advantageously, when a gel is formed, a biologically active compoundpresent in the polymeric solution becomes trapped in the gel structure.This arrangement is conducive to slow release of the biologically activemolecule from the gel structure within a physiological environment.

A biologically active molecule for incorporation into the copolymer orcopolymer composition may be any which causes a physiological change oreffect, such as a low molecular weight compound, drug, antibody, growthfactor, peptide, oligonucleotide, genetic sequence, or compounds thatmodulate cell behaviours such as adhesion, proliferation or metabolism.A biologically active molecule may be attached to the copolymer orincluded in the copolymer composition in order to promote the viabilityor proliferation of cells encapsulated in such gels, or to influence theproduction of compounds by such cells.

The Solvent. Various solvents may be used with the copolymercomposition. The solvent may be aqueous, including water, sodiumchloride solutions such as physiological saline, cell culture media, orany medium that approximates a biological system, such as extracellularmatrix. The pH, and tonicity of a solvent may be any which allowsadjustment as appropriate, so that the environmental condition can beadjusted within the copolymer composition in order for the compositionto take on a gel form. Non-aqueous solvents may be used, or combinationsolvents including a polar organic and an aqueous component. Forexample, an alcohol may be used as the solvent, with or without water.Ethanol, methanol, isopropyl alcohol and other alcohols may be used as asolvent. Other polar organic solvents may be used alone or incombination with water. Non-polar organic solvents may be used withappropriate copolymers, such that A is soluble in the solvent, and B issoluble under certain environmental conditions and insoluble under otherenvironmental conditions.

The term “solvent” may also refer to any prepared mixture of componentswhich may include proteins, growth factors, buffers, ions, and otherco-solutes. For example, culture media and extra cellular solutionscontain water in combination with a number of co-solutes which areconsidered part of the solvent. Further, other soluble components, suchas polymers may be included in the solvent. Such polymers may, forexample, be synthetic polymers or copolymers that do not aggregate withthe copolymer having A(B)n architecture. The solvent may contain, forexample, the polymer used as core component (A) in the copolymer A(B)n.When such a polymer or copolymer is included in the solvent, it wouldnot be considered in the calculation of n_(avg) unless it had astructure A(B)n and was capable of aggregation with arms B of theinventive copolymer. As an example of solvents which include polymers,PEG homopolymer and others may be included in the solvent.

Regardless of the solvent selected for use with the invention, the core(A) is selected to be soluble in the solvent over a range ofenvironmental conditions of interest. The arms (B) areenvironment-responsive components which are soluble in the solvent underone set of environmental conditions, and which become insoluble in thesolvent under different environmental conditions of interest.

Within the composition, the copolymer can be present in the solvent atany concentration that allows gelation to occur, for example a level offrom about 5% to about 50% by weight, or from about 10% to about 25% byweight. This concentration depends on the nature of the solvent and thecopolymer.

Applications of the Invention. The invention may be used for eitherphysiological or industrial applications. Physiological and clinicalapplications of the invention include, but are not limited to, deliveryof biologically active molecules, tissue and biomedical engineering, andtherapeutics. Industrial applications of the invention include but arenot limited to synthetic processes requiring timed release of reactivecomponents, or as barriers.

The invention can be applied to delivery of biologically activemolecules, for example but not limited to in vitro formation of drugdelivery systems, in situ drug delivery, in situ gene delivery. Theinventive polymer may be used to form drug delivery systems in vitro,which could then be implanted into a physiological region of a subject.Drug delivery systems may be formed in situ by suspendingdrug-containing particles in the copolymer composition, then injectingthe composition into, or applying the composition onto specified sitesof a subject causing gel formation to occur in vivo. Genes may bedelivered in vivo using the inventive polymers and compositions. Genedelivery systems in situ can be formed by suspending gene-containingvesicles in the polymer solutions, then injecting the solutions into, orapplying the solutions onto specified sites of patients causing gelformation to occur in vivo. Possible sites for implantation for in vitroformed systems or for insertion of in situ forming systems ofbiologically active molecules include but are not limited to periodontalcavities, intramuscular sites, subcutaneous sites, tumors, bones,joints, intraocular sites, sites that have been exposed by surgery, andwound sites.

The process for forming an in vitro implant may additionally involvemaintaining the composition at least at a gelling temperature prior toinsertion of said implant into a subject, so that the implant does notconvert back to a liquid state.

For compositions having an LCST between ambient temperature and bodytemperature, the environmental condition that triggers gel formation isheating to body temperature. Thus, inserting the composition into thebody causes the biologically active molecule to be trapped in the gel atthe site of application, and sustained release from the site would thenresult.

Further, the invention may be used for in vitro or in situ encapsulationof cells. For encapsulation of cells in vitro, cells can be grown inincubation medium to which the copolymer is added when desirable, so asto keep cells in suspension under certain environmental conditions, butto retain them in a gel when environmental conditions are changed.Encapsulation of cells may also occur in situ by suspending cells in thecopolymer composition under conditions at which the composition is aliquid (for example, below LCST), then injecting the composition into,or applying the composition onto specified sites of patients causing gelformation to occur in vivo. The sites for in situ injection of suspendedcells in the composition, or for insertion of an in vitro formed implantof encapsulated cells can be selected from, but are not limited to,periodontal cavities, intramuscular sites, subcutaneous sites, tumors,bones, joints, intraocular sites, sites that have been exposed bysurgery, and wound sites.

For applications involving encapsulated cells, the length of chainsegments between the physical crosslinks of the copolymer may beselected such that the mesh size between crosslinks provides theappropriate molecular weight cut-off to provide immunoisolation of theencapsulated cells from the intended host while allowing the diffusionof desired nutrients to the cell, and the release of desired agents fromthe encapsulated cells to the host. In an application of in situ formingcell-containing gels, the copolymer would be soluble in water at ambientconditions (ie. room temperature), and the composition includingsuspended cells is injected into or applied onto a patient at thedesired site. Body temperature triggers gel formation, thus causing thecells to be trapped in the gel at the site of injection or application.Cell proliferation and secretion of desired substances from the cell maythen occur.

In cell-containing applications, it may be particularly advantageous toincorporate into the gel peptides or growth factors that promote celladhesion, cell proliferation or otherwise influence cell metabolism inthe desired manner. Such compounds may either be covalently linked tothe copolymer, or incorporated in solid particles or liquid dropletsthat are co-encapsulated in the composition with the cells.

The composition may be used as a coating, barrier, sealant, filling orblocker of an anatomical structure or region, formed either in situ, orformed in vitro and implanted to an appropriate region. The compositionmay be positioned within a biological structure or on top of abiological structure. For example, the composition may be sprayed onto awound site to provide a protective coating for the wound. It may also beinjected into a blood vessel to block blood flow in that vessel. Such anapplication may be useful upstream of a tumor to block blood flow to thetumor. It may also be used as a temporary sealant during surgery.

For physiological applications of the composition according to theinvention, it is advantageous that the gelation is reversible. Forexample, an implant placed in a subject in situ can be reversed byliquifying the implant, such as by localized cooling of the area inwhich the implant was applied. Applying a cold compress, ice, or usingother methods of localized cooling could be used to effect liquificationof the composition from a gel state.

Industrial (non-physiological) applications of the invention includeseparation processes, chemical synthetic processes requiring timed orenvironmentally cued release, or for partitioning of reactants. Forexample, a reactant in an aqueous reaction may be encapsulated withinthe composition in gel form (ie. at a temperature above LCST). When thereaction is cooled below LCST, the encapsulated reactants are releaseddue to the phase change of the composition from gel to liquid, therebyreleasing the encapsulated reactant to the reaction. Thermocyclingreactions which require accurately timed additions of a reactant canincorporate the reactant in the inventive composition to ensure accuraterelease of a reactant at a particular temperature. In other industrialapplications, the copolymer composition can provides a barrier, coating,blockage, sealing or filling. The gelation of the composition formedaccording to the invention is advantageously reversible over a number ofcycles. This reversibility allows repeated gelation and liquificationcycles.

EXAMPLES

Examples of the invention are presented below to illustrate theinvention, but not to limit the scope of the invention.

Reagent Preparation and Handling. Reagents used throughout the examplesare described below, along with appropriate storage requirements.

Ce⁴⁺ Solutions (0.4 M) are prepared by directly dissolving solid cericammonium nitrate Ce(NO₃)₆(NH₄)₂ in distilled deionized water. Thesolution is either prepared fresh everyday, or if it is to be stored fora short duration, it is first sonicated to remove dissolved oxygen, thenplaced in a tightly capped high density polyethylene (HDPE) orpolypropylene (PP) bottle, and stored at 5° C.

NaOH solution (1 N) is prepared from 10 N NaOH by dilution withdistilled deionized water. The prepared solution is stored in a HDPEbottle.

N-isopropyl acrylamide (NIPAAm) monomer of 99% purity stabilized with0.1% methoxyhydroquinone (MHQ), purchased from ACROS Organics, isfurther purified before use. The three major impurities are acrylamidemonomer, MHQ and acrylic acid. They are removed by recrystalizationfollowed by ion-exchange processes. NIPAAm monomer is first dissolved in50/50 heptane/toluene solvent at 60° C. slightly above the melting pointof NIPAAm. The warm solution is filtered through 0.8 μm nylon membraneto eliminate undissolved impurities. The warm aliquot is then put intoan ice bath to recrystalize NIPAAm monomer. The NIPAAm crystal isrecovered by vacuum filtration. The recrystalization process is repeatedtwice to eliminate MHQ and residual acrylamide monomers. The solid isthen dissolved in distilled deionized water as a 20 wt % solution. Thesolution is poured into a bed of anionic exchange resin (IRA-402, Cl⁻form, SUPELCO) to eliminate trace amount of acrylic acid. The anion-freeNIPAAm solution is separated from the resin by vacuum suction. The highpurity NIPAAm monomer is then recovered by freeze-drying at −55° C., andunder a vacuum below 10⁻⁴ bar.

OH-terminated PEG is selected and obtained as follows. Polyethyleneglycol of various architectures and with varying number of chain endsterminated by reactive hydroxyl groups are purchased from ShearwaterPolymers, Inc. They are used without further purification ormodification.

The composition of the standard extracellular solution (formulated forbeta-cell lines) was as follows: NaCl (140 mM), KCl (4 MM), MgCl₂ (1mM), CaCl₂ (2 mM), and HEPES (10 mM). The final pH of 7.3 was achievedby adjusting with NaOH.

F-12K Nutrient Mixture (Kaighn's Modification) made by GibcoBRL was usedas the cell culture media referred to herein as F-12K. Purified collagenused in any methods herein was Vitrogen™, obtained from CohesionTechnologies Inc., Palo Alto, Calif.

Example 1 Two-Armed Block Copolymer PNIPAAm-PEG-PNIPAAm

Linear polyethylene glycol (MW 5077) with terminal hydroxyl groups atboth ends of the chain, HO(CH₂CH₂O)₁₁₃H, was purchased from ShearwaterPolymer (product name Sunbright DKH-50H, Lot. 68559) and used withoutfurther treatment. This reagent is herein referred to as the two-armedPEG.

The two-armed PEG (1.0 g) was mixed with 1.35 g of purified NIPAAm,dissolved in water, then mixed with 2.0 ml of a 0.4 M Ce⁴⁺ solution, and0.8 ml of 1N NaOH solution. The total mixture was 15 ml in volume. Thereagents were cold mixed at 5° C. and sonicated to eliminate dissolvedgas. The reaction was then allowed to proceed at 30° C. for 24 hours. Atthe end of 24 hours, the mixture was diluted to 100 ml by adding colddistilled water and placed in a 5° C. refrigerator to quench thereaction. The entire reaction was carried out under a helium blanket.The reaction vial was made of polypropylene instead of glass to avoidCe⁴⁺/OH-glass side reaction, which could lead to an increased productionof PNIPAAm homopolymer. The product of this reaction is predominantly atri-block copolymer consisting of a central segment of PEG, with twoseparate segments of PNIPAAm covalently attached to either end of thePEG segment.

The unreacted NIPAAm, PEG and residual Ceric salts, and PNIPAAmhomopolymer were removed by dialysis using an ester cellulose membrane(Fisher Scientific) in a water bath for four weeks. The water waschanged every 24 hours. The copolymer was recovered from the solution byhigh vacuum freeze-drying at −55° C.

A 10 wt % sample was prepared by dissolving one gram of the tri-blockpolymer in 9.0 ml of cold water at 5° C. Below 30° C., the solution wascolorless and transparent. Between 5° C. to 25° C., the solution was lowin viscosity and thus could be easily drawn into a syringe through a 25gauge needle. Upon heating to above 32° C., the solution became opaqueimmediately, and the entire 10 mL solution turned into a solid white gelin less than two minutes. The gel occupied the entire solution volume.The gel showed some elasticity, and could hold its own shape even whenthe sample vial was inverted. Storage at 37° C. resulted in slightshrinkage of the gel (10% in 24 hours, 20% in two months). Differentialscanning calorimetry measurements of the sample showed an endothermicfirst order transition temperature at 33.1° C. The width at half peakheight was 2.2° C. The phase transition observed was completelyreversible over many cycles.

Example 2 Four Armed Block Copolymer PEG-(PNIPAAm)₄

Four-arm branched polyethylene glycol (MW 10486) with one terminalhydroxyl group at each branch was purchased from Shearwater Polymer(product name Sunbright PTE-10000, Lot. 76606) and used without furthertreatment. This reagent is herein referred to as the 4-armed PEG.

The four-armed PEG (1.0 g) was mixed with 1.35 g of purified NIPAAm,dissolved in water, then mixed with 2.0 ml of a 0.4 M Ce⁴⁺ solution, and0.8 ml of 1N NaOH solution. The total mixture was 15 mL in volume. Thereagents were cold mixed at 5° C. and sonicated to eliminate dissolvedgas. The reaction was then allowed to proceed at 30° C. for 24 hours. Atthe end of 24 hours, the mixture was diluted to 100 ml by adding colddistilled water and placed in a 5° C. refrigerator to quench thereaction. The entire reaction was carried out under a helium blanket.The reaction vial was made of polypropylene instead of glass to avoidCe⁴⁺/OH-glass side reaction, which could lead to an increased productionof PNIPAAm homopolymer. The product of this reaction is predominantly abranched copolymer consisting of a central four-armed PEG, andindividual PNIPAAm segments covalently attached to the end of each armof the four-armed PEG.

The unreacted NIPAAm, PEG and residual Ceric salt, and PNIPAAmhomopolymer were removed by dialysis using an ester cellulose membrane[Fisher Scientific] in a water bath for four weeks. The water waschanged every 24 hours. The copolymer is recovered from the solution byhigh vacuum freeze-drying at −55° C.

A 10% wt sample was prepared by dissolving one gram of the four-armedcopolymer in 9.0 ml of 5° C. cold water. Below 30° C., the solution wascolorless and transparent. Between 5° C. to 25° C., the solution was lowin viscosity and thus could be easily drawn into syringe through a 25gauge needle. Upon heating to above 32° C., the solution became opaqueimmediately, and the entire 10 mL solution quickly turned into a solidwhite gel in less than two minutes. The gel occupied the entire solutionvolume. The gel showed some elasticity. It could hold its own shape evenwhen the sample vial was inverted. The gel was cohesively strong enoughto be picked up by a pair of tweezers, and was stronger than the gelformed according to Example 1. Storage at 37° C. resulted in negligibleshrinkage (less than 5% in two months). Differential scanningcalorimetry measurements of the sample showed an endothermic first ordertransition temperature at 32.6° C. The width at half peak height was3.4° C. The phase transition observed was completely reversible overmany cycles.

Example 3 Eight Armed Block Copolymer PEG-(PNIPAAm)₈

Eight-arm branched polyethylene glycol (MW 19770) with a terminalhydroxyl group at each branch was purchased from Shearwater Polymer(product name Sunbright HGEO-20000, Lot. 7D543) and used without furthertreatment. This reagent is herein referred to as the 8-armed PEG.

The eight-armed PEG (1.0 g) was mixed with 1.35 g of purified NIPAAm,dissolved in water, then mixed with 2.0 ml of a 0.4 M Ce⁴⁺ solution, and0.8 ml of 1N NaOH solution. The total mixture was 15 mL in volume. Thereagents were cold mixed at 5° C. and sonicated to eliminate dissolvedgas. The reaction was then allowed to proceed at 30° C. for 24 hours. Atthe end of 24 hours, the mixture was diluted to 100 ml by adding colddistilled water and placed in a 5° C. refrigerator to quench thereaction. The entire reaction was carried out under a Helium blanket.The reaction vial was made of polypropylene instead of glass to avoidCe⁴⁺/OH-glass side reaction, which can lead to an increased productionof PNIPAAm homopolymer. The product of this reaction is predominantly abranched copolymer consisting of a central block of the eight-armed PEG,and eight separate segments of PNIPAAm covalently attached to the end ofeach arm of the eight-armed PEG.

The unreacted NIPAAm, PEG and residual Ceric salt, and PNIPAAmhomopolymer were removed by dialysis using ester cellulose membrane[Fisher Scientific] in a water bath for four weeks. The water waschanged every 24 hours. The copolymer is recovered from the solution byhigh vacuum freeze-drying at −55° C.

A 10 wt % sample was prepared by dissolved one gram of the eight-armedcopolymer product in 9.0 ml of cold water at 5° C. Below 30° C., thesolution was colorless and transparent. Between 5° C. to 25° C., thesolution was low in viscosity and thus could be easily drawn intosyringe through a 25 gauge needle. Upon heating above 32° C., thesolution became opaque immediately, and the entire 10 mL solutionquickly turned into a solid white gel in less than two minutes. The geloccupied the entire solution volume. The gel showed some elasticity. Itcould hold its own shape even when the sample vial was inverted. The gelwas cohesively strong enough to be picked up by a pair of tweezers, andwas stronger than the gel formed according to Example 1, and comparablein strength to the gel of Example 2. Storage at 37° C. resulted innegligible gel shrinkage (less than 5% in two months). Differentialscanning calorimetry measurements of the sample showed an endothermicfirst order transition temperature at 33.5° C. The width at half peakheight was 2.8° C. The phase transition observed was completelyreversible over many cycles.

Example 4 Synthesis, Purification and Thermal Characteristics of 50/50Copolymers of PEG/PNIPAAm having Architecture A(B)₂, A(B)₄ and A(B)₈

The copolymers were synthesized by Ce⁴⁺/OH redox initiated free radicalpolymerization in water. Four hydroxyl-terminated PEGs were purchasedfrom Shearwater and used without further purification: monomethoxy-PEGof 2,000 Da (i.e., 1 arm of length 2,000 Da), linear PEG diol of 4,600Da (i.e., 2 arm PEG with each arm length of 2,300 Da), 4 arm star PEG of9,300 Da (arm length=2,325 Da), and 8 arm star PEG of 19,700 Da (armlength=2,460 Da). All functionalized PEGs have polydispersity indices ofless than 1.04.

The following exemplary conditions and procedure may be used for batchsynthesis of PEG-PNIPAAm copolymers having architecture A(B)₂, A(B)₄ andA(B)₈, shown for comparison purposes with AB. The reaction solutionvolume is 30 ml in all cases. No NaOH is added to the reaction solution.The solvent is distilled water, and the reaction temperature is 30° C.The reagents are cold mixed at 5° C., and then sonicated for 10 to 20minutes to eliminate dissolved oxygen. Subsequently, the mixture issubjected to an inert gas surge for 5 minutes to pre-saturate thesolution with Helium gas. The reaction is then allowed to proceed for 24hours under a water-saturated Helium blanket. The reaction vessel ismade of Teflon (or polypropylene) instead of glass to avoidCe⁴⁺/OH-glass side reaction, which could lead to an increased productionof homopolymer. At the end of 24 hours, the mixture was diluted to 100ml by adding cold distilled water and placed in a 5° C. refrigerator toquench the reaction. The detailed reaction conditions are summarized inTable 1. TABLE 1 Conditions for Batch Synthesis of 50/50 PEG/PNIPAAmCopolymers 0.4 M PEG/ Reaction Reaction Reaction Ce⁴⁺ NIPAAm PEG PEG/PNIPAAm Temp. volume Duration vol. mass mass NIPAAm final Structure (°C.) (ml) (hr) (ml) (g) (g) feed ratio composition AB 30 30 24 8.0 1.0 3.0 75/25 49/51 A(B)₂ 30 30 24 8.0 0.81 1.5 65/35 48/52 A(B)₄ 30 30 248.0 0.87 1.2 58/42 51/49 A(B)₈ 30 30 24 8.0 0.79 1.0 56/44 49/51

The copolymers were purified by dialysis. Cellulose ester membrane ofvarious molecular weight cut-off [Fisher Scientific] were selected forsuch purpose. The details of purification conditions are provided inTable 2, including the molecular weight cut off (MWCO) of dialysis tubesused, and the recovered yield calculated as (dry copolymer)/(initial PEGmass+initial monomer mass). TABLE 2 Conditions for Purification of 50/50PEG/PNIPAAm Copolymers MWCO of Dialysis Recovered Copolymers Tube TimeYield (wt %) AB 3,500 4 weeks 15˜25% A(B)₂ 8,000 4 weeks 20˜30% A(B)₄15,000 4 weeks 20˜30% A(B)₈ 25,000 4 weeks 15˜25%

The copolymer molecular weights were determined by proton NMR [VarianUnity Plus 500 MHz]. The ratio of the methyl protons in isopropyl groupsto the methylene protons of PEGs was used to determine the ratio ofNIPAAm to ethylene glycol repeat units. Using the known molecular weightof PEG, the molecular weight of PNIPAAm segments, and thus the copolymermolecular weight can be deduced. The composition and characteristics ofcopolymers formed are given in Table 3. TABLE 3 Composition andCharacteristics of Copolymers “A” Block PEG MW Weight^(a) per Arm “B”Block PEG/PNIPAAm Total Molecular Structure (Da) (Da) Weight (Da)^(b,c)(by weight) Weight (Da)^(c) AB 2,000 2,000 2,100 ± 200 49/51 4,100 ±200   A(B)₂ 4,600 2,300 2,500 ± 200 48/52 9,600 ± 400   A(B)₄ 9,3002,330 2,200 ± 200 51/49 18,200 ± 800   A(B)₈ 19,700  2,460 2,600 ± 20049/51 40,000 ± 1,600 ^(a)As reported by manufacturer; polydispersity of 1.04 or better.^(b)Average from three synthesis batches^(c)As calculated from NMR analysis

The thermal characteristics of the copolymers were determined bydifferential scanning calorimetry (DSC) [TA2010, TA Instrument). DSCscans of aqueous solutions of each copolymer at various concentrationswere taken at a heating rate of 2° C./minute. Transition temperatures,both onset of thermal transitions (T_(onset)) and peak temperature ofendotherm (T_(max)), and the enthalpy of thermal transition normalizedto PNIPAAm content, ΔH (J/g of PNIPAAm, were determined. The results aretabulated in Tables 4 to 7 for copolymers, and in Table 8 for solutionsof PNIPAAm homopolymer (comparative example) in water. The measurementprecision for temperature is ±0.2° C. and enthalpy is ±2 J/g for allcases. TABLE 4 DSC Results for 1 arm 50/50 PEG/PNIPAAm Copolymer AB(Comparative Example) ΔH (J/g of Concentration T_(onset) (° C.) T_(max)(° C.) PNIPAAm) 20% 27.3 28.7 30 15% 28.7 29.8 32 10% 30.0 31.0 35

TABLE 5 DSC Results for 2 arm 50/50 PEG/PNIPAAm Copolymer A(B)₂ ΔH (J/gof Concentration T_(onset) (° C.) T_(max) (° C.) PNIPAAm) 20% 26.4 28.529 15% 28.6 30.0 36 10% 30.0 31.0 38

TABLE 6 DSC Results for 4 arm 50/50 PEG/PNIPAAm Copolymer A(B)₄ ΔH (J/gof Concentration T_(onset) (° C.) T_(max) (° C.) PNIPAAm) 20% 26.2 29.329 15% 29.4 30.9 34 10% 30.2 31.4 37

TABLE 7 DSC Results for 8 arm 50/50 PEG/PNIPAAm Copolymer A(B)₈ ΔH (J/gof Concentration T_(onset) T_(max) PNIPAAm) 20% 28.2 30.3 28 15% 29.731.1 33 10% 30.8 32.0 34

TABLE 8 DSC Results for PNIPAAm homopolymer (Comparative Example) ΔH(J/g of Concentration T_(onset) (° C.) T_(max) (° C.) PNIPAAm) 10% 32.633.6 43 7.5%  32.8 33.7 44  5% 32.7 33.4 45

The results show that the transition temperature is concentrationdependent. As concentration decreases, the transition temperature risesslightly. The range of onset temperature is between 26° C. to 31° C. forall four types of copolymers, which is a suitable range for aphysiological application requiring a liquid state at an ambienttemperature and a gel state at a physiological temperature. The range ofΔH values illustrates that the copolymer molecular architectureinfluences the phase transition of the PNIPAAm segments, while thecomparison between the copolymers and the homopolymer suggests that thepresence of PEG may have prevented PNIPAAm segments from fullycollapsing. The enthalpy of gelation for copolymers according to theinvention are about 15% to 35% lower than that of PNIPAAm homopolymer(see Table 8) measured at the equal PNIPAAm content.

Example 5 Rheological Properties and Gelation Mechanism of Block andStar Copolymers of PEG and PNIPAAM of Varying Architectures

Block or star copolymers with a central hydrophilic polyethylene glycol(PEG) segment as core (A), and temperature responsivepoly(N-isopropylacrylamide) (PNIPAAm) terminal segments as arms (B) ofvarious architectures A(B)₂, A(B)₄ and A(B)₈, were synthesized toinvestigate the structures and properties relationship. A comparativecopolymer having the structure AB is also evaluated. The synthesis andpurification of copolymers were conducted according to the schemes givenin Example 4. The compositions of the copolymers are identical to thosegiven in Example 4 (see Table 3, Composition and Characteristics ofCopolymers). All four copolymers evaluated herein are of approximately50/50 PEG/PNIPAAm ratio by weight.

At 5° C., the viscosities of 20% wt solutions were between 700 to 950cP, and they could be easily injected through a 25G needle. Upon warmingto body temperature, A(B)₂, A(B)₄ and A(B)₈ formed a strong associativenetwork gel with aggregates of PNIPAAm segments acting as physicalcrosslinks, whereas AB formed a weaker gel by micellar packing andentanglement. The values of elastic modulus, loss tangent, and yieldstrength were between 1300 to 2600 Pa, 0.4 to 0.6, and 300 to 1000 Pa,respectively.

The mechanical and rheological properties of the copolymers werecharacterized using a temperature controlled rheometer (Carri-Med, TAInstrument] with a cone and plate (4 cm diameter, 2 degree angle)geometry. Yield stress (σ_(c)), critical strain (γ_(c)), and elastic andloss moduli (G′, G″) were determined under oscillatory mode at 37° C.Solution viscosities were measured under flow mode at 5° C. using 20 wt% copolymer solutions in water.

FIG. 3 illustrates a composition according to the invention comprisingthe A(B)₄ polymer of PEG and PNIPAAm in aqueous solution at aconcentration of 20% by weight. As shown in picture A, the compositionis a liquid at room temperature (25° C.), and forms a strong gel at bodytemperature (37° C.), as shown in picture B.

FIG. 4 illustrates that for a 20% solution of A(B)₄, the onset of bothelastic and loss modulus, shown as (A) temperature sweep of oscillatorymeasurement, is between the onset and the peak temperatures in theendotherm as detected by (B) DSC temperature scan. When PNIPAAmcollapses at an elevated temperature, heat is evolved, and measured byDSC. The endotherm of a DSC scan is corresponding to the molecular eventof PNIPAAm segments collapsing. The synchronization of the endotherm andmoduli onset temperatures illustrates that the thermal transitions arelinked to the mechanical changes, and therefore the aggregation measuredby the thermal transitions are at least partially intermolecular innature. It is known that the mechanical strength (i.e., modulus) isscale to the number of crosslinks per unit volume. The higher thecrosslink density, the higher the modulus will be. The inter-aggregationwill lead to connection between molecules (i.e., forming physicalcrosslinks) which ultimately gives rise to a high mechanical strength;whereas, intra-aggregation will produce no physical crosslinks, thus nodrastic rise of modulus is expected upon transition temperature. Therapid rise of modulus at the onset of endotherm illustrates a gelationmechanism of network formation for the multiple arm copolymers.

FIG. 5 shows the superposition of DSC scans for multiple cycles at 2°C./min, for aqueous compositions comprising either (A) the eight-armedpolymer A(B)₈ or (B) the four-armed polymer A(B)₄, both at aconcentration of 20% wt in water. All samples were subjected to cyclicheating and cooling for up to 30 cycles. The thermal behaviour of thematerial is completely reversible. There was a small hysteresis of 2° C.observed between heating and cooling curves.

The superposability of the scans indicates that the gelation process iscompletely reversible. Although the enthalpy of gel melting is identicalto the enthalpy of gel formation, at a heating/cooling rate of 2°C./min, there is a difference between the peak temperatures of theheating endotherm and the cooling exotherm of about 2° C. in the scansshown in FIG. 5. The difference can be attributed to the kinetics ofgelation process. At infinitely slow cooling/heating rates, the two peaktemperatures should be identical. Another thermoreversible polymerhydroxypropylmethyl cellulose (HPC) has been reported to have atemperature lag of 8 to 110° C. at a much slower heating rate of 0.25°C./min (Sarkar, Journal of Applied Polymer Science, 24: 1073-1087,1979). The relatively small temperature hysteresis seen with thesecopolymers is indicative of rapid gelation kinetics compared to that ofcellulose derivatives.

FIG. 6 illustrates the Theological behavior of copolymers at bodytemperatures. The viscoelastic and mechanical properties were evaluatedby subjecting the gels at 37° C. to oscillatory stresses (σ) that rangedfrom 0.1 to 3,000 Pa at 1 Hz frequency, and measuring the resultingstrains (γ). The elastic modulus (G′), loss modulus(G″), overall modulus(G*=[G′²+G″²]^(1/2)) and loss tangent, (tan δ≡G″/G′) were thencalculated.

The elastic modulus of A(B)₂, A(B)₄ and A(B)₈ copolymers are between1,300 to 2,600 Pa, which makes them “hard gels”, and tan delta valuesare between 0.4 to 0.7, which indicate that they are very much“solid-like”. The examples provided in FIG. 6 are compositions of A(B)₂,A(B)₄ and A(B)₈ copolymers at 20% wt in water. A rheological scan of theone-arm copolymer (AB) composition (20% wt in water) is also provided asa comparative example. Multiple arm copolymers in general have a highermodulus than a single arm copolymer. A higher modulus means a highernumber of load bearing chains per unit volume; and that is believed tobe due to the very nature of having multiple aggregation blocks in onemolecule. A one-arm copolymer is also different from the multiple armcopolymers in terms of its yielding behavior. Passing the yield point,the tan delta increases as stress increases, whereas the multiple armcopolymers behave otherwise. This behavior suggests a different gelationmechanism. For compositions comprising 20% wt copolymer in water, A(B)₄showed the highest modulus and highest yield stress, and is thus thestrongest material among the four polymers prepared.

FIG. 6, part A and B show that for all four gels, G′ and G*(respectively) are approximately constant over a wide range ofoscillatory stress values, then decrease sharply at high stress. Thestress at which the decrease occurs is different among the four gels.

Table 9 provides a summary of the constant G′ and G″ values atlinear-viscoelastic region, which shows that G′ and G″ increase as thenumber of arms increase from 1 to 2 to 4, but then decrease upon furtherbranching to an 8 arm architecture. The G′, G″ values shown in Table 9were measured at ω=1 Hz, and σ=50 Pa which is within the linearviscoelastic region of all the materials above. Values are the averagesfrom three synthesis batches. Accordingly, loss tangent for all fourgels is also seen to be approximately constant over a wide range ofoscillatory stress values, but then deviate from linearity at highstress, as illustrated in FIG. 6, part C. The yield point is defined aswhere overall modulus, G*, deviates from linearity as illustrated inFIG. 6, part B. The corresponding stress and strain are called criticalyield stress and strain. TABLE 9 Gel Strength of Copolymers at 37° C.G′, G″ at linear viscoelastic region σ_(c), γ_(c), Critical Materials G′(Pa) G″ (Pa) Loss tan G″/G′ Yield Stress (Pa) Yield Strain AB  630 ± 130180 ± 30 0.28 ± 0.03 750 ± 90 1.10 ± 0.15 A(B)₂ 2100 ± 200 850 ± 80 0.40± 0.04 600 ± 70 0.30 ± 0.03 A(B)₄ 2600 ± 250 1400 ± 150 0.53 ± 0.04 1000± 150 0.37 ± 0.04 A(B)₈ 1300 ± 150 800 ± 90 0.62 ± 0.04 300 ± 50 0.22 ±0.03

The gelation mechanism suggests that multiple PNIPAAm segments arerequired in the same molecule in order for physically crosslinkedhydrogel networks to be formed. Thus it is expected that the two-,four-, and eight-arm structures would form gels via a physicalcrosslinking mechanism, while the one-arm diblock copolymer would form agel via the micellelar aggregation mechanism. Comparison of therheological results for the two-, four- and eight-arm structures showsthat the elastic and loss moduli in the linear viscoelastic region, aswell as the yield stress and strain are all highest for the four-armstructure, indicating that the four-arm copolymer forms gels that arehighest in strength, as well as deformability. Branching should have twoeffects on gel rheology. Increasing the number of arms should increasethe degree of crosslinking in the gel via the covalent linkage of arms;hence gel strength should increase. However, as the number of armsincreases, aggregation between PNIPAAm blocks within the same moleculebecomes increasingly favored over inter-molecular aggregation. Sinceintramolecular aggregation does not contribute to physical crosslinking,degree of physical crosslinking would decrease as branching increasesbeyond a certain point. The maximum in gel strength observed for the4-arm gel may thus be explained by the counterbalancing effects ofcovalent crosslinking and physical crosslinking. It is also interestingto note that the loss tangent increases monotonically from 0.40 to 0.53to 0.62 as the degree of branching increases from two to eight arms,indicating that the relative viscous component increases with the degreeof branching.

A comparison of the rheological behavior of the one-arm micellaraggregate gels to the multi-arm physically crosslinked gels shows thatthe most striking difference between the two classes of gels is that theloss tangent decreases at high stress for the one-arm gel while for allthe other gels, loss tangent increases at high stress. The contrast intrends is suggestive of a fundamental difference in the gel structure.The viscous component of the one-arm gel becomes increasingly dominantat high values of oscillatory stress, while the elastic component of themulti-arm gels become increasingly dominant.

The one-arm gel shows a significantly lower values of G′ than themulti-arm gels. According to Hvidt's classification, (Hvidt, et al.,Journal of Physical Chemistry, 98:12320-12328, 1994; Almgren, W Brown,S. Hvidt, Colloid and Polymer Science, 273:2, 1995), the one arm gelwould be considered a “soft gel” (i.e., G′<1,000 Pa), while the otherswould be considered “hard gels” (i.e., G′>1,000 Pa). In contrast to thelow modulus, the one arm gel has the highest critical strain among thegels, and a relatively high yield stress, lower than only the four-armgel. With only one end tethered to PNIPAAm aggregates, PEG segments inone arm gels are more freely mobile and more readily deformable than PEGsegments in multi-arm gels that are tethered at both ends. The lowmodulus and high critical strain of one-arm gel are a reflection of theease of deformability. Likewise, with only one end sterically shieldedby PNIPAAm aggregates, the free end of PEG segments in one-arm gels areallowed to interact with other PEG segments and form entanglements. Therelatively high yield stress of one-arm gels may be the result ofsignificant entanglement of PEG corona.

The viscosity of PEG-PNIPAAm copolymer solutions was measured at 5° C.,and for a shear rate range of 0.1 to 200 s⁻¹. For a shear rate greaterthan 5 s⁻¹, all solutions are essentially Newtonian. The viscosity for20% wt a one-arm diblock, two-arm triblock, four-arm star and eight-armstar are 750 cP, 950 cP, 900 cP and 700 cP respectively. All thesesolutions are of low enough viscosity to easily inject through a 25Gneedle.

This example illustrates that block and star copolymers of PEG andPNIPAAm form liquid aqueous solutions at low temperature, and transformto relatively strong elastic gels upon heating. Multiple arm copolymersform gels via a physical crosslinking mechanism, while diblockcopolymers gel by a micellar aggregation mechanism. The rheologicalproperties of the gels are dependent on the molecular architecture, withA(B)₄ showed optimal properties (i.e., at 20% wt). The copolymercompositions according to the invention show relatively low injectionviscosities and high gel strengths, and are therefore useful forclinical and physiological applications such as in situ drug delivery,cell encapsulation and anatomical barriers.

Example 6 Toxicity of Eight-Arm PEG/PNIPAAm Copolymer

The toxicity of a the eight-arm copolymer of PEG and PNIPPAm was testedin F-12K culture media using HIT insulinoma cells (INS-1). Solutionshaving 1%, and 3% concentrations of the eight-arm copolymer in culturemedium were tested. F-12K culture medium included 10% fetal bovineserum. The control (0%) solution was prepared as F-12K culture medium(with 10% fetal bovine serum), but without copolymer. Multi-well plates(0.5 mL/well) were seeded with HIT cells, and either acopolymer-containing solution or the control culture medium. The wellswere examined for cell viability up to 50 hours;

These dilute copolymer solutions showed no effect on cell viability interms of % dead cells and total number of live cells compared to thecontrol medium. The eight-arm PEG-PNIPAAm copolymer illustratedcompatibility with the HIT insulinoma cells for incubations up to 50hours. The copolymer was evaluated for toxicity at low levelconcentrations in culture medium so that all copolymer molecules wouldbe freely accessible to cells. Cells would be less exposed to thepolymer molecules when in gel form (ie- at higher copolymerconcentrations) and it would be expected that cell toxicity of the gelform of the polymer would be considerably less than that of thedissolved form of the polymer.

Example 7 Gelation Phase Conversion for PEG/PNIPAAm CopolymerCompositions

The copolymer compositions prepared according to the invention take on agel form at different temperatures depending on a number of parameters.A four-arm copolymer prepared according to Example 4 was examined forgelation in different solvents.

Gelation phase diagram observations were made using both a visual methodand an inverted tube method. The gelation temperature is defined to bethe temperature at which the composition (polymer/solvent mixture)becomes completely opaque.

Further, the inverted tube method was used to assess gelation point.Using a 1.4 cm round diameter tube, a composition is defined to be inthe gel state if it does not flow after the tube has been inverted for10 seconds. The gelation temperatures determined using the visual endpoint and inverted tube methods were identical.

For cell culture media or extracellular solution media, at polymerconcentrations of less than about 14 wt %, compositions became turbidsuspensions of white solid particles upon heating that flow easilyinstead of gelling. Thus, at concentrations lower than 14 weightpercent, compositions of polymers in these media do not form gels. Inwater, no gel forms below 6 wt % polymer, and in 157 mM saline solution,no gel forms below 7 wt % polymer.

The gelation temperature increases as the concentration of copolymer inthe composition decreases. Standard extracellular solution depresses theLCST. The polymer is less viscous in this solvent compared to the othersolvents tested.

FIG. 7 shows the phase diagrams of compositions comprising the copolymerin different solvents: (a) water, (b) physiological NaCl (157 mM), (c)F-12K Media, and (d) standard extracellular solution. The diagrams showtemperature/concentration conditions at which the compositions(polymer/solvent mixtures) exist as solutions or gels, as well as theminimum concentration required for gel formation to occur. These dataillustrate that the nature of the solvent affects the temperature atwhich a gel forms. A variety of different concentrations shown in thephase diagram would be appropriate for clinical and/or physiologicalapplications of the composition.

Example 8 Copolymer of Nitrocellulose

A copolymer according to the invention is formed using nitrocellulose asarms (B) and PEG as core (A). A copolymer having either A(B)₄ or A(B)₈architecture is formed. A gelable composition according to the inventioncomprises dilution of the nitrocellulose/PEG copolymer in ethanol at aconcentration of about 10%.

Nitrocellulose in ethanol forms a gel upon warming. The gelationtemperature depends on molecular weight and concentration. For amolecular weight of 197,000, the gelation temperature is 10° C., 5.5° C.and −20° C. for a polymer fraction of 0.5%, 1% and 4% respectively, andthe theta temperature was found to be 301-310 K (Newman et al., J. Phys.Chem. 60:648-656, 1955).

Example 9 Composition Comprising Different Copolymers

A composition according to the invention is formed using copolymershaving AB and A(B)₄ architecture, as described above in Example 4. Thecomposition comprises a total of 15 wt % copolymer in physiologicalsaline. The proportion of AB to A(B)₄ in the composition is 2:3,resulting in an average n value (n_(avg)) of 2.8 (or 14/5). Theresulting composition is liquid at ambient temperature, and converts toa gel when injected into a subcutaneous site of a subject. This changefrom liquid to gel is due to a change in environmental condition,specifically the change from ambient temperature to body temperature.

Example 10 Composition Comprising Different Copolymers

A composition according to the invention is formed using a graftcopolymer having A′(B)₃ architecture and a block copolymer having A″(B)₄architecture, as described above in Example 4. In this case A′ differsfrom A″. Each A is a PEG of differing molecular weight. However, B isthe same (PNIPAAM) for both types of copolymer. The compositioncomprises a total of 13 wt % copolymer in cell culture media. Theproportion of A′(B)₃ to A″(B)₄ in the composition is 10:3, resulting inan average n value (n_(avg)) of 3.23. The resulting composition isliquid at ambient temperature, and converts to a gel at a temperaturebelow body temperature.

The above-described embodiments of the present invention are intended tobe examples only. Alterations, modifications and variations may beeffected to the particular embodiments by those of skill in the artwithout departing from the scope of the invention, which is definedsolely by the claims appended hereto.

1. A gelable composition comprising a copolymer and a solvent, thecopolymer having the structure A(B)n, wherein A is soluble in thesolvent, B is convertible between soluble and insoluble in the solventdepending on an environmental condition, and n is greater than 1, thecomposition being convertible from liquid to gel under an environmentalcondition where B is insoluble.
 2. The composition according to claim 1,wherein the environmental condition is selected from the groupconsisting of temperature, pH, ionic strength, and a combinationthereof.
 3. The composition according to claim 2, wherein theenvironmental condition is temperature.
 4. The composition according toclaim 1, 2 or 3, wherein A is selected from the group consisting ofpolyethylene glycol (PEG), polyvinyl pyrrolidone, polyvinyl alcohol,polyhydroxyethylmethacrylate, and hyaluronic acid.
 5. The compositionaccording to any one of claims 1 to 4, wherein B is selected from thegroup consisting of poly-N-isopropyl acrylamide (PNIPAAm),hydroxypropylmethyl cellulose and other methyl cellulose derivatives,poly(ethylene glycol vinyl ether-co-butyl vinyl ether), polymers ofN-alky acrylamide derivatives, poly(amino acid)s, peptide sequences,poly(methacryloy L-alanine methyl ester), poly(methacryloy L-alanineethyl ester) and nitrocellulose.
 6. The composition according to any oneof claims 1 to 5, wherein the copolymer is present in the solvent at alevel of from 5% to 50% by weight.
 7. The composition according to claim6 wherein the copolymer is present in the solvent at a level of from 10%to 25% by weight.
 8. The composition according to claim 1, wherein n is2, 4, or
 8. 9. The composition according to claim 1, wherein n greaterthan or equal to
 4. 10. The composition according to claim 8 or 9,wherein A is polyethyleneglycol (PEG).
 11. The composition according toany one of claims 8 to 10, wherein B is poly-N-isopropyl acrylamide(PNIPAAm).
 12. The composition according to any one of claims 8 to 11,wherein the solvent is aqueous.
 13. The composition according to claim12, wherein the solvent is selected from the group consisting of water,physiological saline and cell culture media.
 14. The compositionaccording to any one of claims 1 to 13, additionally comprising abiologically active molecule.
 15. The composition according to any oneof claims 1 to 14, additionally comprising an architecturally differentcopolymer, wherein n_(avg) is greater than
 1. 16. The compositionaccording to claim 15, wherein said architecturally different copolymeris AB.
 17. A gelable copolymer having the structure A(B)n, wherein A issoluble in a desired solvent, B is convertible between soluble andinsoluble in the desired solvent depending on an environmentalcondition, n is greater than 1, and A(B)n forms a gel in the solventunder an environmental condition where B is insoluble.
 18. The copolymeraccording to claim 17, wherein the environmental condition is selectedfrom the group consisting of temperature, pH, ionic strength, and acombination thereof.
 19. The copolymer according to claim 18, whereinthe environmental condition is temperature.
 20. The copolymer accordingto any one of claims 17 to 19, wherein A is selected from the groupconsisting of polyethylene glycol (PEG), polyvinyl pyrrolidone,polyvinyl alcohol, polyhydroxyethylmethacrylate, and hyaluronic acid.21. The copolymer according to any one of claims 17 to 20, wherein B isselected from the group consisting of poly-N-isopropyl acrylamide(PNIPAAm), hydroxypropylmethyl cellulose and other methyl cellulosederivatives, poly(ethylene glycol vinyl ether-co-butyl vinyl ether),polymers of N-alky acrylamide derivatives, poly(amino acid)s, peptidesequences, poly(methacryloy L-alanine methyl ester), poly(methacryloyL-alanine ethyl ester) and nitrocellulose.
 22. The copolymer accordingto any one of claims 17 to 21, wherein n is 2, 4, or
 8. 23. Thecopolymer according to claim 17, wherein A is polyethyleneglycol (PEG).24. The copolymer according to claim 17, wherein B is poly-N-isopropylacrylamide (PNIPAAm).
 25. The copolymer according to any one of claims17 to 24, additionally comprising a degradable linker (C) between A andB.
 26. The copolymer according to claim 17, wherein A is degradable. 27.The copolymer according to any one of claims 17 to 26, additionallycomprising a biologically active compound (D) bound to component A or B.28. A gelable copolymer comprising a core selected from compoundssoluble in a desired solvent, and arms selected from compoundsconvertible between soluble and insoluble in the desired solventdepending on an environmental condition, wherein the copolymer forms agel in the solvent under an environmental condition where the arms areinsoluble.
 29. The copolymer according to claim 28, additionallycomprising a biologically active compound.
 30. The copolymer accordingto claim 28 or 29, additionally comprising a degradable linker betweenthe core and at least one arm.
 31. The copolymer according to claim 29,additionally comprising a degradable linker joining the biologicallyactive compound to the core.
 32. The copolymer according to claim 29,wherein the core is degradable.
 33. A gelable composition comprising acopolymer according to any one of claims 17 to 32 in combination withthe desired solvent.
 34. The gelable composition according to claim 33,wherein said desired solvent includes a polymer.
 35. An in situ formingimplant comprising a gelable composition according to claim 1, whereinthe solvent is aqueous and the environmental condition comprises heatingto a temperature between ambient temperature and body temperature.
 36. Aprocess for forming a gelable composition comprising the steps of: (i)forming a copolymer having the structure A(B)n, wherein A is soluble ina solvent of interest, B is convertible between soluble and insoluble inthe solvent depending on an environmental condition, and n is greaterthan 1; (ii) solubilizing said copolymer in the solvent in an amountadequate to convert the composition from liquid to gel under anenvironmental condition where B is insoluble.
 37. A process of in situimplant formation comprising the steps of (i) forming a gelablecomposition according to claim 1, wherein said solvent is aqueous, andsaid environmental condition comprises heating to a temperature betweenambient temperature and body temperature and (ii) inserting saidcomposition into a subject.
 38. A process for forming an implant invitro comprising the steps of (i) forming a gelable compositionaccording to claim 1, wherein said solvent is aqueous and saidenvironment condition comprises heating to a gelling temperature betweenambient temperature and body temperature, and (ii) heating saidcomposition to at least said gelling temperature.
 39. The processaccording to claim 38, additionally comprising the step of maintainingsaid composition at least at said gelling temperature prior to insertionof said implant into a subject.